Method of detecting colon cancer

ABSTRACT

An immunological assay and kit for colon cancer screening is disclosed. Fecal glycoproteins are extracted from individual samples such that immunogenicity is maintained. The purified fecal glycoproteins are reacted with antibodies to Colon and Ovarian Tumor Antigen (COTA). The mucin antigen COTA is specifically present in colorectal cancer tissue and not in normal colons. The amount of COTA in the fecal sample is determined and used to indicate the presence of colon cancer.

FIELD OF THE INVENTION

The present invention is directed to a method for detecting colon cancerutilizing an antibody specific for a glycoprotein found in colon cancercells.

BACKGROUND OF THE INVENTION

Colorectal cancer is among the leading causes of cancer-relatedmorbidity and mortality in industrialized nations. The pathogenesis isrelated to hereditary influences, modified by the quantity and qualityof dietary fat. In 1995, the American Cancer society estimated that135,000 new cases of colon cancer were diagnosed; 71% were in the colonand 30% were in the rectum. Patients diagnosed at an early stage, priorto lymph-node spread, are potentially cured with surgery. At present,only 41% of patients are diagnosed at an early stage. The remainingcases frequently undergo peri-operative radiation and/or chemotherapy toattempt to control the metastatic spread of disease. Ultimately, 50% ofpatients thought to have undergone curative resections eventuallydevelop recurrent disease. Unfortunately, 55,000 Americans die each yeardue to recurrent or metastatic colon or rectal cancer. The key toenhanced survival is early diagnosis. Colon and rectal cancers are oftensilent and slowly progressive. Most patients exhibit symptoms such asrectal bleeding, pain, abdominal distension or weight loss only afterthe disease is advanced and not surgically curable.

Over the past 25 years, early colorectal cancer detection has been basedon the fecal occult blood test (FOBT) performed annually on asymptomaticindividuals. Current recommendations adapted by several healthcareorganizations, including the American Cancer Society, call for fecaloccult blood testing beginning at age 50, repeated annually until suchtime as the patient would no longer benefit from screening. A positiveFOBT leads to colonoscopic examination of the bowel; an expensive andinvasive procedure, with a serious complication rate of one per 5,000examinations. Only 12% of patients with heme positive stool arediagnosed with cancer or large polyps at the time of colonoscopy. Moststudies show that FOBT screening does not improve cancer-relatedmortality or overall survival. Compliance with occult blood testing hasbeen poor; less than 20percent of the population is offered or completesFOBT as recommended. If FOBT is properly done, the patient collects afecal sample from three consecutive bowel movements. Samples areobtained while the patient adheres to dietary guidelines and avoidsmedications known to induce occult gastrointestinal bleeding. Inreality, physicians frequently fail to instruct patients properly,patients frequently fail to adhere to protocol, and some patients findthe task of collecting fecal samples difficult or unpleasant, hencecompliance with annual occult blood testing is poor. Compounding theproblem of compliance, the sensitivity and specificity of FOBT to detectcolon cancer is poor. In eight prospective studies where hemocculttesting was followed by colonoscopy, only 41 of 159 cancers diagnosedwere detected by FOBT, yielding a screening sensitivity of 26%. FOBTsensitivity for pre-cancerous polyps was also poor. Poor testspecificity leads to unnecessary colonoscopy, adding considerableexpense to colon cancer screening. In the University of Minnesota trial,a large prospective hemoccult screening study, test specificity was 90%,and positive predictive value was 2%. Only one colon cancer was found inevery 50 test-triggered colonoscopies performed.

New methodology of immunological testing has potential advantages overFOBT including improved sensitivity, specificity and patient compliance.If immunological testing is more sensitive and specific than FOBT, thefrequency of testing could be reduced, collection of consecutive sampleswould be eliminated, dietary and medication schedule modifications wouldbe eliminated, and patient compliance would be enhanced. If colon cancerscreening by immunological testing is more specific, the problem offalse positive test results leading to unnecessary colonoscopicexamination would be reduced leading to cost savings and improvedsafety. Clearly, there is a long felt need for a simple, accurate, andinexpensive screen for colon cancer.

DESCRIPTION OF THE RELATED ART

Since the goblet cells of colorectal cancers produce glycoproteinmucin(s) that are immunologically distinguishable from normal colonicmucin (Nairn et al. Br. Med. J. 1791-1793, 1962) it is possible todetect their presence in the feces by immunological assay. Springer(Springer Science 1198-1206, 1984) reported that T antigens (ThomsenFriedenreich), and Tn antigens, precursors of MN blood groupglycoproteins, are tumor associated antigens. Kurosaka et al (Kurosakaet al J. Biol. Chem. 258: 11594-11598, 1983) isolated severaloligosaccharides from a rectal adenocarcinoma and reported that one ofthe major oligosaccharides was sialylated-Tn or STn (NeuAcα2-6GalNAcα1-o-ser/thr). Ovine submaxillary mucin is identical in chemicalstructure as STn disaccharides. Kjeldsen et al. (Kjeldsen, et al. CancerRes. 48: 2214-2220, 1988) produced TKH1 and TKH2 monoclonal antibodiesthat react with ovine submaxillary mucin and demonstrated that both werereactive by immunohistochemistry with adenocarcinoma of human lung,stomach, colon, breast and pancreas, but not with normal human tissuesamples. Itzkowitz et al. (Itzkowitz et al. Cancer Res. 260: 8262-8271,1989) observed expression of all the three Tn, Sialosyl-Tn and Tantigens in colon cancers. He proposed that in malignancy incompleteglycolsylation and early sialylation of precursor antigen results inpremature termination of the carbohydrate chain. In contrast, Podolsky(Podolsky J. Bio. Chem. 260: 15510-15515, 1985) had isolated 21oligosaccharides from normal colonic mucin and sialylated-Tn was one ofthem. The non-reactivity of TKH2 antibody with normal colonic mucosa wasexplained by the work of Jass et al. (Jass et al. Pathology 26: 418-422,1994) and later Ogata et al. (Ogata et al. Cancer Res. 55: 1869-1874,1995) who reported that in the normal colon sialic acid is heavilyo-acetylated and is masked, thus antibodies cannot react, while inneoplastic tissue o-acetylation is not extensive, and sialic acid is notmasked. Removal of the O-acetyl groups from normal colon tissue byalkaline treatment made them reactive with TKH2 antibody.

Pant et al. did initial studies on Colon-Ovarian tumor antigen (COTA)with polyclonal antibodies produced against mucin extracted from humancolon cancer tissue removed at surgery. The polyclonal antibodies weremade specific by absorption with lyophilized extracts of normal humancolon and other normal human tissues and CEA. The resultant antibodyretained immunoreactivity towards colon cancer and mucinous ovariantumors but did not react with normal colon tissue as seen byimmunodiffusion and immunofluorescence testing (Pant et al. Tumor Biol.5: 243-254, 1984). Furthermore, the absorbed anti-COTA antibodiesimmunostained several colon cancer tissues and LS174T tumor cells andother colon cancer xenografts but did not stain normal human colonsections (Pant et al. Am J. Clin. Path. 86:1-9, 1986). Pant used COTAisolated from LS174T tumor cells to produce a monoclonal antibody SP-21.This antibody gave identical immunohistochemical staining pattern asobserved with absorbed polyclonal anti-COTA antibodies (Pant et al.Hybridoma 5: 129-135, 1986). In an extended immunohistochemical studywith SP-21 monoclonal antibody, Dorman et al (Dorman et al. J. Clin.Path. 45: 932-933, 1992) observed that SP-21 immunostained several otherhuman cancer tissues including ovary, stomach, breast, esophagus,prostate, pancreas and endometrium.

The relationship of COTA to STn was established by chemical analysis ofpurified COTA, blocking of immunoreactivity by N-acetyl neuraminic acidand loss of immunoreactivity after neuraminidase treatment. It wasconcluded that neuraminic acid is an essential component of COTA (Pant,et al. Journal of Tumor Marker Oncology 3:1-13, 1988). The identity ofCOTA to STn was further established by comparison of SP-21 with TKH1.Kordari et al. reported neuraminidase treatment and O-glycanasetreatments of colon cancer tissue completely destroyed TKH1 and COTAepitope reactivity indicating that TKH 1 and COTA monoclonal antibodiesrecognized the NeuAcα2-6GalNAc disaccharide exclusively (Kordari, et al.Tumor Biol. Abs., 1990).

SUMMARY OF THE INVENTION

The present disclosure is drawn to a method for extracting glycoproteinsfrom a fecal sample such that immunogenicity is maintained comprisingthe steps of:

(a) obtaining a fecal sample from an individual;

(b) shaking the fecal sample in a preservative solution;

(c) separating the solution containing the fecal sample to produce afraction comprising glycoproteins;

(d) precipitating the glycoproteins from the fraction comprisingglycoproteins; and

(e) dissolving the precipitated glycoproteins in buffer.

The method may further comprise the steps of:

(f) centrifuging the solution from step (e) to produce a pellet and asupernatant; and

(g) collecting the supernatant containing the extracted glycoproteins.

In a preferred embodiment, the fecal sample is collected in a clean vialcontaining preservative wherein the preservative comprises ethanol andformalin at a concentration such that bacterial growth is retarded andextraneous fecal matter is precipitated while maintaining immunogenicityof glycoproteins in the fecal sample. Preferably, the preservativecomprises 25-45% ethanol with 0.025%-0.35% formalin. More preferably,the preservative comprises 40% ethanol with 0.25% formalin.

In a preferred embodiment, the solution containing the fecal sample isseparated by centrifugation. More preferably, the centrifugation is at1040-1500×g for 10-15 minutes at room temperature.

In a preferred embodiment, the glycoproteins are precipitated from thefraction comprising glycoproteins with 3 volumes of 100% ethanol with0.1 ml of 20% sodium acetate. More preferably, the, precipitationproceeds for about 3 hours at room temperature. Preferably, theprecipitated glycoproteins are dissolved in phosphate buffered saline.

The present disclosure is also drawn to a method for screening for coloncancer comprising:

(a) obtaining purified fecal glycoproteins, said glycoproteins beingobtained by a method comprising:

(i) obtaining a fecal sample from an individual;

(ii) shaking the fecal sample in a preservative solution;

(iii) separating the solution containing the fecal sample to produce afraction comprising glycoproteins;

(iv) precipitating the glycoproteins from the fraction comprisingglycoproteins; and

(v) dissolving the precipitated glycoproteins in buffer; and

(b) determining the level of COTA antigen in the purified fecalglycoproteins.

In a preferred embodiment, the fecal sample is collected in a clean vialcontaining preservative wherein the preservative comprises ethanol andformalin at a concentration such that bacterial growth is retarded andextraneous fecal matter is precipitated while maintaining immunogenicityof glycoproteins in the fecal sample. Preferably, the preservativecomprises 25-45% ethanol with 0.025%-0.35% formalin. More preferably,the preservative comprises 40% ethanol with 0.25% formalin.

In a preferred embodiment, the solution containing the fecal sample isseparated by centrifugation. More preferably, the centrifugation is at1040-1500×g for 10-15 minutes at room temperature.

In a preferred embodiment, the glycoproteins are precipitated from thefraction comprising glycoproteins with 3 volumes of 100% ethanol with0.1 ml of 20% sodium acetate. More preferably, the precipitationproceeds for about 3 hours at room temperature. Preferably, theprecipitated glycoproteins are dissolved in phosphate buffered saline.

In a preferred embodiment, the determination of the level of COTAantigen in the purified glycoproteins comprises the steps of:

(a) reacting an antibody for COTA antigen with the extractedglycoproteins to form a complex of the antibody and the COTA antigen;

(b) exposing the complex to a second antibody, wherein said secondantibody is a detection agent; and

(c) determining the level of the detection agent and in turn determiningthe presence of COTA antigen in the fecal sample.

In one embodiment, the antibody for COTA antigen is bound to a solidsurface. In an alternate embodiment, the extracted glycoproteins arebound to a solid surface. Preferably, the antibody for COTA antigen ismonoclonal antibody SP-21.

The present disclosure is also drawn to a kit for screening for coloncancer comprising:

an anti-COTA capture antibody bound to a solid support;

purified human COTA antigen; and

a vial containing a preservative solution.

In one embodiment, the solid support is an ELISA plate. In an alternateembodiment, the solid support is a membrane filter. In a preferredembodiment, the kit contains the monoclonal antibody SP-21 as theantibody for COTA antigen.

In a preferred embodiment, the kit contains a preservative solutionwhich comprises 25-45% ethanol with 0.025%-0.35% formalin. In a mostpreferred embodiment, the kit contains a preservative comprising 40%ethanol with 0.25% formalin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plot of optical density of slot-dots obtained on dilutions ofpurified COTA protein from LS 174T.

FIG. 2. Lane A. Graded intensity of slot-dots of purified COTAdilutions. Lane B. Slot-dots of 3 patients with colon cancer, 1 withulcerative colitis and dysplasia, 1 with polyp, 3 normal individuals andcontrol PBS.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The hemocult test is based on pseudoperoxidase activity of heme whichoxidizes gum guaiac to a blue color. It is well recognized that the testalso detects presence of blood in the digestive tract from sources otherthan colorectal cancers or adenomas. Furthermore, the peroxidaseactivity is also present in some foods. Certain chemicals which inhibitthe pseudoperoxidase emzyme activity can yield false negative resultsaffecting the sensitivity and specificity of the test.

The present disclosure relates to an immunological assay method and kitto apply to population-based colon cancer screening which avoidsproblems associated with the hemocult test discussed above.

In a preferred embodiment, the fecal sample is obtained using acommercially available stool collection device such as one availablefrom Ability Building Center, Rochester, Minn., and is then placedimmediately in a preservative solution so as to inhibit bacterialmultiplication and sample glycoprotein degradation. Glycoproteins arethen isolated from the fecal sample at a later time. The preservativesolution contains ethanol and formalin at a concentration such thatbacterial growth is retarded and extraneous fecal matter is precipitatedwhile maintaining immunogenicity of glycoproteins in the fecal sample.Extraneous fecal matter includes components other than the glycoproteinfraction. Preferably, the preservative solution contains 0.025%-0.35%formalin in 25-45% ethanol. In a preferred embodiment, the preservativecontains 40% ethanol with 0.25% formalin to optimally prevent bacterialmultiplication and glycoprotein degradation without loss of glycoproteinimmunoreactivity.

A key feature of the presently claimed invention is the extraction ofthe desired glycoproteins from the preservative solution whilemaintaining immunogenicity. Any method for extraction of glycoproteinswhich maintains immunogenicity of the extracted mucins may be used inthe screening method. In general, the fecal sample in the preservativesolution is separated to obtain a fraction containing the glycoproteins.Any means known to those skilled in the art may be used to fractionatethe fecal sample including, but not limited to centrifugation,filtration, and chromatography. In a preferred embodiment,centrifugation is used to isolate the glycoprotein-containing fraction,preferably at 1040×g to 1500×g for 10-15 min. Most preferably,centrifugation at 1040×g for 10 minutes at room temperature is used toobtain a supernatant containing glycoproteins. Theglycoprotein-containing fraction is then further purified byprecipitation of the glycoproteins including COTA. Means to precipitatethe glycoproteins include, but are not limited to, precipitation withammonium sulfate, or organic solvents, such as ethanol and acetone, or acombination thereof. Alternatively, the glycoproteins may be furtherpurified by means known to those skilled in the art such as columnchromatography, gel filtration and the like. In a preferred embodimentthe glycoprotein-containing fraction is precipitated with ethanol andsodium acetate and then centrifuged to obtain a precipitate. Theprecipitated pellet is resuspended in buffer and recentrifuged to obtaina supernatant containing the glycoproteins to be screened, preferably at1500×g to 2040×g for 15-20 minutes. In a preferred embodiment, theresuspended pellet is centrifuged at 1500×g for 15 minutes.

The level of COTA antigen in the supernatant containing theglycoproteins is then determined. The mucin antigen COTA is specificallypresent in colorectal cancer tissue and not in normal colon tissue.Since COTA mucin is produced by goblet cells and eventually gets mixedwith the feces, detection and estimation of it in the fecal samples isthe basis of the present non-invasive test for colorectal cancer.

Any antibody which binds to Colon and Ovarian Tumor Antigen (COTA) maybe used for quantitation of COTA levels as a colon cancer screen. Arepresentative monoclonal antibody includes SP-21. A monoclonal antibodywhich binds to COTA may be obtained by isolation of COTA from the LS174Tcell line (ATCC CL-188) using well known methods (Kohler and Milstein(1975) Nature 256: 495-497, for example). The antibody for COTA antigenis reacted with the extracted glycoproteins to form a complex of theantibody and the COTA antigen. In a preferred embodiment, a combinationof one IgG antibody and one IgM antibody is used. Any means availablefor facilitation of antibody-antigen binding may be used in thedisclosed method including but not limited to tubes, filters, beads,multiwell plates and a mixture thereof. Preferred embodiments use eitherELISA plate technology or slot dot assays. Means to quantitate theextent of binding include detection using colorimetric assays as well asradioimmunoassay. In a preferred embodiment, the complex of the antibodyand the COTA antigen is exposed to a second antibody which is labelledsuch that the level of COTA antigen in the glycoprotein sample may bedetected and quantitated by reference to a standard curve prepared fromdilutions of purified COTA. Such labels include, but are not limited to,radioactive and colorimetric methods including absorption,bioluminescence and fluorescence labeling means. In a preferredembodiment, the second antibody is biotinylated and is subsequentlytreated with peroxidase conjugated streptavidin to produce aquantifiable colorimetric signal. Levels of COTA antigen detected by theDot assay greater than 15 μg/ml predict the presence of colon cancerwith sensitivity of 83% and specificity of 96%. ELISA methodology mayalso be used to detect COTA antigen. A cut off value for detection ofcolon cancer in μg/ml will be based upon values obtained from normalindividuals.

The invention is further set forth in the following examples, which arein no way intended as a limitation upon the scope of the invention.

EXAMPLES

A more detailed description of the present invention is provided below.While the described embodiment represents the preferred embodiment ofthe present invention, it is to be understood that modifications willoccur to those skilled in the art without departing from the spirit ofthe invention. The scope of the invention is therefore to be determinedsolely by the appended claims.

Example 1

Patient Selection and Sample Collection

One hundred patients with risk factors or signs of colon cancerscheduled for colonoscopic examination were contacted. Indications forcolonoscopy included hemoccult positive stools, rectal bleeding, priorhistory of adenoma or colon cancer, or family history of colon cancer. Akit containing a vial with 20 ml of preservative (40% ethanol with 0.25%formalin solution) and a fecal collection device were mailed to thepatient. Patients were asked to collect approximately 1-2 gm of fecesand place it into the vial. The kit was returned on the day of theircolonoscopy.

Extraction of Glycoproteins From Fecal Samples

The contents of the vial were shaken vigorously to mix the fecal samplecompletely and then centrifuged at 1040×g for 10 minutes at roomtemperature (RT) to remove solid fecal debris and the clear supernatantwas collected. 3 volumes of 100% ethanol with 0.1 ml of 20% sodiumacetate was added to the supernatant containing the glycoproteinfraction. Precipitation proceeded for 3 hours at room temperature. Theprecipitate was collected by centrifugation at 1040×g for 10 minutes.Traces of ethanol from the precipitate were removed by aeration. Theprecipitate was dissolved in 1 ml of Phosphate Buffered Saline (PBS).The preparation was further centrifuged at 1500×g for 15 minutes and theclear supernatant was collected.

Protein Quantitation

Protein quantitation of the samples was done by Pierce Bicinchoninicreagent. The assay contained (a) 100 μl each of doubling dilutions ofbovine serum albumin (BioRad) ranging from 745 μg/ml, 372 μg/ml, 186μg/ml, 93 μg/ml and 46.5 μg/ml for obtaining a standard curve; (b) 100μl of a sample of purified COTA; and (c) 100 μl of PBS as negativecontrol. 2 ml of complete BCA reagent was added and tubes were coveredand incubated at RT for 2 hours. O.D. of the sample was taken at 562 nmin a SHIMADZU UV-160 spectrophotometer. All patient samples were dilutedand normalized to 60 μg/ml protein concentration. Alternatively, proteinconcentrations can be obtained by spectrophotometry by adding 1 ml ofsample to a quartz cuvette and measuring absorbance at 280 nm.

Standard Curve of COTA

Highly purified COTA was prepared from LS174T tumors (ATCC #CL-188) byWestphals hot phenol method for complex carbohydrates (Westphal et al.Zeitschrift Fur Naturforschung 76: 148-155, 1952). Briefly, the mucinoustumor was treated with equal volume of phenol and heated at 65 C for 30minutes. After cooling and centrifugation, the upper water solublefraction was separated and precipitated with 3 volumes of ethanol. Theprecipitate was dissolved in PBS, pH 7.4, dialysed and recentrifuged. Itwas fractionated on a Sepharose-4B column and the fractions containingCOTA reactivity were pooled and concentrated. The preparation containedvery low protein and had high COTA reactivity. Two-fold dilutions ofCOTA ranging in protein values of 90 μg/ml, 45 μg/ml, 22.5 μg/ml, 11.2μg/ml and 5.6 μg/ml were used in the dot assay and in preparation of thestandard curve.

Colonoscopy

Patients were prepped for diagnostic colonoscopy by adhering to a clearliquid diet the day prior to exam, and taking an oral bowel preparation(COLYTE) the night prior to exam. Patients underwent colonoscopy, thegold standard diagnostic test for colorectal neoplasia, with OLYMPUS orPENTAX video endoscopy, by board-certified gastroenterologists appointedto the GI laboratories of Loma Linda University Medical Center or LomaLinda Veterans Administration Medical Center. Diagnostic information,including the size and location of any neoplastic lesion was recorded inthe procedure note, which in-turn was forwarded to the GI researchlaboratory for data acquisition. The written pathology report wasobtained from the pathology Department, LLUMC or LLVAMC, and informationwas abstracted regarding tumor stage. If the patient underwent surgicalresection, the operative report and final pathology report were reviewedto obtain tumor stage.

Slot-Dot Assay of Samples and of Dilutions of Purified COTA

0.2 μm PROTAN nitrocellulose membrane (Schleicher and Schuell) wasplaced in a Bio-Rad slot-dot apparatus. 20 μl of each test sample, knownpositive and negative control samples, and doubling dilutions ofpurified COTA and PBS were applied in the slots. The samples wereallowed to bind to the membrane for 30 minutes. The membrane was thenblocked with 3% non-fat milk for 30 minutes. The membrane was washedwith PBS three times and then reacted with 1 μg/ml of monoclonal SP-21antibody for 60 minutes in a humid chamber at RT. The nitrocellulosestrip was then washed with PBS and reacted with 1:2 diluted biotinylatedgoat anti-mouse antibody (DAKO LSAB2 kit) and incubated for 15 minutesat RT in a humid chamber. After washing three times with PBS, themembrane was reacted with peroxidase conjugated streptavidin (DAKOLSAB2) for 10 minutes at RT in a humid chamber. The membrane was washedthree times with PBS and then reacted with a solution of 5 mg/10 ml of3,3′ diaminobenzidine tetrahydrorhloride made in Tris buffer, pH 7.2with 0.01% H₂O₂ for 90 seconds. The membrane was washed with running tapwater and allowed to dry. The intensity of the slot-dot color wasquantitated by reflectance reading in a Bio-Rad model GS-700 imagingdensitometer and computer generated O.D. values were obtained. Astandard curve was drawn by plotting the COTA protein versus the opticaldensity values. From this curve, corresponding COTA equivalent proteinvamounts of the samples were calculated. Samples showing protein amountsgreater than 15 μg/ml for COTA were interpreted as positive.

Example 2

Screening for Detection of Colon Cancer Using Slot Blot Assay

On application of 20 μl of doubling dilutions of 90 μg/ml of purifiedCOTA in slot-dot assay, a graduation in color intensity was obtained(FIG. 2, lane A). Plotting the densitometer reading of each slot-dot ofpurified COTA dilution against the protein amount (Table 1) resulted ina curve with 99.8% coefficient of correlation (FIG. 1). Samples withO.D. value exceeding 0.45 (15 μg/ml COTA protein) were consideredpositive. Positive slot-dots on 3 colon cancer patients, 1 ulcerativecolitis and 1 polyp and negative slot-dots on 2 normal colon extractsand PBS with values lower than 15 μg/ml are seen in FIG. 2, lane B.

TABLE 1 Densitometer readings of slot-dots of purified COTA Adjustedvolume COTA protein (μg/ml) O.D. (mm × mm) 90 3.09 45 1.56 22.5 0.6111.2 0.21 5.6 0.074

Sample kits were sent to 123 patients scheduled for colonoscopicexamination. 94 patients returned the sample for analysis (76.4%).Examination of 100 fecal samples for COTA levels (Table 2) consisted of94 individuals who were examined by colonoscopy and 6 healthy volunteerswho did not have colonoscopy. Of the 6 patients diagnosed with coloncancer, 5 had COTA levels higher than 15 μg/ml thus resulting in 83.3%positivity. The mean COTA value of 6 colon cancer patients was 20.8μg/ml and the mean value of 58 normal patients was 7 μg/ml. Theseresults indicate 83.3% sensitivity and 96.5% specificity of the test. Ofthe 6 patients who had prior colorectal cancers, 4 had resection of thetumors and 2 had radiation treatment of rectal cancer. Of these withpreviously treated colon cancer, one patient was positive but had 6 mmsessile polyp and had colonic obstruction at the time the fecal COTAtest was performed. Of 22 cases of polyps with polyp size ranging from 2mm to 25 mm, 6 had values over 15 μg/ml thus resulting in 27.2%positivity. Of 8 ulcerative colitis cases tested, one patient wasstrongly positive. Colonoscopy with biopsy on this patient revealed highgrade dysplasia. The group of normal patients included 6 healthy normalvolunteers. Of the remaining 52 patients who had colonoscopy done,findings include: 21 normal, 26 with diverticulosis, 4 with hemorrhoidsand one with anal fissure. A single case of hemorrhoids and anal fissuregave values in the positive range thus resulting in 3.44% positivity.These results indicate that large percentage of patients with colorectalcancer can be detected when screened for COTA in their feces.

It may be of interest to periodically follow the patients who hadearlier radiation treatment or surgery or had polyps and were stronglypositive for COTA. It is of significance that one of the ulcerativecolitis patients who had very high amounts of COTA in the feces was alsodiagnosed with high grade dysplasia (see Table 2, below). Amongindividuals who had normal colonoscopy, only 3.4% had COTA levels higherthan 15.0 μg/ml. This indicates specificity and suitability of the testfor screening colorectal neoplasms.

TABLE 2 Patient samples showing COTA values of 15 μg/ml or higher aspositives Patients No. tested No. positive No. negative % positiveColorectal CA 6 5 1 83.3 Colorectal CA 6 1 5 16.6 (Resected or radiationtreated) Polyps 22 6 16 27.2 Ulcerative 7 0 7 0 colitis Ulcerative 1 1 0100 colitis with dysplasia Normals 58 2 56 3.4

Example 3

Hybridoma Production

Monoclonal anti-COTA producing hybridomas were generated by immunizationof Balb/cj mice with highly purified COTA followed by fusion ofsplenocytes with SP2/0 myeloma cells. Hybridoma fluids of IgG isotypeclones 13B5, 9B4, 15C11 and 15H11 and IgM isotype clones 2C3, 3G4 andIBH2 have shown inmunoreactivity towards COTA. The monoclonal antibodies13B5 (IgG) and 2C3 (IgM) will be further purified by columnchromatography and protein A binding. Other monoclonal antibodies ofhybridomas (9B4 and 15C11 or 15H11) produced in our laboratory and notyet tested will be analyzed to determine if ELISA system sensitivity andspecificity can be enhanced.

Example 4

ELISA Assay for the Detection of COTA

ELISA for quantification of COTA in stool extracts will be developed.The ELISA will be validated for crude stool extracts and purified stoolfractions. Twenty-four samples (in duplicate) can be processed in asingle ELISA plate. Details of ELISA plate development are as follows:

13B5 (IgG) (or alternate antibody) will be bound in the wells of amicrotiter plate as the “capture” antibody. Patient fecal samplesappropriately purified and diluted as described above will be added tothe wells. If COTA is present in the fecal sample, it is immobilized bybinding to the capture antibody. Bound COTA is then detected by theaddition of horseradish peroxidase conjugated monoclonal antibody 2C3(IgM) (or alternate antibody), and subsequent addition ofO-phenylenediamine-H₂O₂ made in citrate buffer and taking the O.D. ofthe color reaction at 492 nm. The amount of COTA in each sample isdetermined by comparison with a standard curve of COTA protein serialdilutions.

For each ELISA test plate, positive standards will include ovinesubmaxillary mucin and purified human COTA antigen in sufficientquantity as part of the ELISA kit. The standards developed will betested in a protein matrix similar to the final sample preparationprovided in the kit. Alternate ELISA configuration will be formulatedand tested. This will include binding of COTA antigen directly to theELISA 96-well plate and detecting its presence by 13B5 (IgG) and 2C3(IgM) monoclonal antibodies. To test this procedure, additional clonesIgG isotypes 9B4, 15C11 and 15H11 and IgM isotypes 3G4 and 18H2 withknown reactivity to COTA will be grown in hybridoma growth tissueculture medium and subsequently purified by liquid chromatography.

Antibody Binding to the Plate and Blocking the Reactive Sites

100 μl of the capture antibody diluted in binding buffer to 10 μg/ml isadded to each well. The plate is then washed 3×with Tris bufferedsaline, pH 7.6, and then blocked by adding 200 μl of SUPERBLOCK to eachwell. The washing procedure is repeated two more times. The microtiterplate is used immediately in ELISA test or alternatively air dried atroom temperature. The dried plates are then sealed in a ziplock plasticbag containing desiccant and stored up to 12 months at 4C.

Antigen Bound to Plates for Testing the Presence of COTA or for TestingHybridoma Fluids

Wash plate 3 times with previously bound antigen and then block theremaining sites with 300 μl of blocking buffer and incubate for 30 min.The plate is then washed 3 times in wash buffer. 100 μl of the firstantibody or test antibody is diluted to proper concentration in carrierbuffer and added to blanks, positive and controls (Hybridoma fluids notto be diluted in carrier buffer). The plates are incubated for 60 min.at room temperature and then washed 3 times with wash buffer. 100 μl ofsecond antibody diluted in carrier buffer is added to each well andincubated for 60 min. The plate is then washed 3 times with wash buffer.Then 100 μl of proper substrate is added to each well and the plate isincubated for 30-60 min. at room temperature. The absorbance is thenread using a plate reader with wavelength set according to type ofenzyme-linked second antibody and substrate used.

Data Analysis

COTA levels expressed in micrograms per ml stool sample will bedetermined by ELISA. Levels will be correlated with the clinical gradeand stage of any neoplasm present, i.e. pre-cancerous polyp,non-invasive or invasive colon cancer, and with the disease location,i.e.: rectosigmoid, descending, transverse or ascending colon. Testsensitivity and specificity will be determined for disease stage andlocation. The positive predictive value of the COTA immunology testcompared with paired hemocult testing will be reported.

What is claimed is:
 1. A method for screening for colon cancer in anindividual comprising: (a) obtaining purified fecal glycoproteins, saidglycoproteins being obtained by a method comprising: (i) obtaining afecal sample for the individual; (ii) shaking the fecal sample in apreservative solution; (iii) separating the solution containing thefecal sample to produce a fraction comprising glycoproteins; (iv)precipitating the glycoproteins from the fraction comprisingglycoproteins; and (v) dissolving the precipitated glycoproteins inbuffer; and (b) determining the level of COTA antigen in the purifiedfecal glycoproteins from the fecal sample, thereby screening for coloncancer in the individual.
 2. The method of claim 1 wherein the fecalsample is collected in a clean vial containing preservative wherein thepreservative comprises ethanol and formalin at a concentration such thatbacterial growth is retarded and extraneous fecal matter is precipitatedwhile maintaining immunogenicity of glycoproteins in the fecal sample.3. The method of claim 2 wherein the preservative comprises 25-45%ethanol with 0.025%-0.35% formalin.
 4. The method of claim 3 wherein thepreservative comprises 40% ethanol with 0.25.% formalin.
 5. The methodof claim 1 wherein the solution containing the fecal sample is separatedby centrifugation.
 6. The method of claim 5 wherein centrifugation is at1040-1500×g for 10-15 minutes at room temperature.
 7. The method ofclaim 1 wherein the glycoproteins are precipitated from the fractioncomprising glycoproteins with 3 volumes of 100% ethanol with 0.1 ml of20% sodium acetate.
 8. The method of claim 7 wherein the precipitationproceeds for about 3 hours at room temperature.
 9. The method of claim 1wherein the precipitated glycoproteins are dissolved in phosphatebuffered saline.
 10. A method according to claim 1 wherein determinationof the level of COTA antigen in the purified glycoproteins comprises thesteps of: (a) reacting an antibody for COTA antigen with the purifiedfecal glycoproteins to form a complex of the antibody and the COTAantigen; (b) exposing the complex to a second antibody, wherein saidsecond antibody is a detection agent; and (c) determining the level ofthe detection agent and in turn determining the presence of COTA antigenin the fecal sample.
 11. The method of claim 10 wherein the antibody forCOTA antigen is bound to a solid surface.
 12. The method of claim 10wherein the extracted glycoproteins are bound to a solid surface.